Low density lipoprotein receptor-related protein 8 (LRP-8) (also called ApoER2) is a cell surface receptor and is a member of the LDL receptor family. LRP-8 is abundant in the brain and placenta. LRP-8 ligands include reelin, ApoE, selenoprotein P and endoplasmic reticulum resident receptor-associated protein (RAP). LRP-8 plays a role in endocytosis, transcytosis and signal transduction, in particular in embryonic neuronal migration and postnatal long-term potentiation. LRP-8 isoform 1 consists of 963 amino acids and is separated into a ligand binding domain of eight ligand binding regions (Accession No: Q14114.4). While all LDL receptor family proteins consist of a cytoplasmic tail, LRP-8 is unique in that it contains a proline-rich 59 amino acid insert encoded by the alternatively spliced exon 19. This insert allows for protein interactions that are unable to occur with other LDL receptors and also diminishes the function of LRP-8 in lipoprotein endocytosis (Myant (2010) Proc. Biol. Sci. 277(1680): 345-51).
Through interactions with one of its ligands, reelin, LRP-8 also plays a critical role in the migration of neurons during development. Another LDL family member, VLDLR, also interacts with reelin, and together these two receptors modulate brain development and function. LRP-8 also functions as a receptor for the cholesterol transport protein apolipoprotein E. Decreased expression of LRP-8 is thus associated with certain neurological diseases. For instance, studies show that manipulation of LRP-8 can lead to Alzheimer's disease. A decrease in LRP8 expression is observed in patients with Alzheimer's disease. LRP-8 synthesis can lead to increased gamma secretase activity, a protease which cleaves LRP-8 as well as amyloid precursor protein (APP) into amyloid β (Aβ), resulting in degrading products that control the expression of a tau protein, which ultimately leads to Alzheimer's disease (Carter (2007) Neurochem Int. 50(1): 12-38). LRP-8 activity has also been linked to antiphospholipid syndrome and major depressive disorder (MDD).
The blood-brain barrier (BBB) is a highly selective permeability barrier formed by brain endothelial cells that separates circulating blood from the brain extracellular fluid. It acts to effectively protect the brain from many common bacterial infections. While the BBB allows for the passage of water, some gases, and selective molecules, the BBB severely limits the penetration of large molecule drugs into the brain. Antibodies are generally too large to cross the BBB, and only certain antibiotics are able to cross. In some cases, a drug must be administered directly into the cerebrospinal fluid. However, drugs delivered directly to the cerebrospinal fluid often do not effectively penetrate into the brain tissue itself.
Several mechanisms have been developed for drug targeting in the brain that involve going “through” or “behind” the BBB. One of the strategies devised to overcome this obstacle includes utilizing transcytosis trafficking pathways of endogenous receptors expressed at the brain capillary endothelium. Recombinant proteins, such as monoclonal antibodies, have been designed against these receptors to enable receptor-mediated drug delivery. Recent studies suggest that antibodies with low affinity to BBB receptors, such as the transferrin receptor (TfR), offer the potential to substantially increase BBB transport and CNS retention of associated therapeutic moieties/molecules compared with high-affinity antibodies. These low affinity antibodies maximize brain uptake while minimizing reverse transcytosis back to the blood and also maximize the extent of accumulation after therapeutic dosing (Atwal et al. (2011) Sci. Transl. Med. 3: 84ra43; Yu et al. (2011) Sci. Transl. Med. 3(84): 84ra44). Yet, the safety of administering such antibodies and conjugates is not well known.
Other BBB receptors used for binding an antibody to mediate transport across the BBB include the insulin receptor, insulin-like growth factor receptor (IGF receptor), LRP-8, low density lipoprotein receptor-related protein 1 (LRP1), glucose transporter 1 (Glutl) and heparin-binding epidermal growth factor-like growth factor (HB-EGF). Antibodies and binding proteins that target some of these receptors and transport therapeutic agents across the BBB have been proposed.
Moreover, engineered proteins, such as antibodies, fragments, and multispecific binding proteins capable of binding two or more antigens, are known in the art. Such multispecific binding proteins can be generated using cell fusion, chemical conjugation, or recombinant DNA techniques. There are a variety of multispecific binding protein structures known in the art and many structures and methods have distinct advantages or disadvantages.
Bispecific antibodies have been produced, for instance, using quadroma technology. Bispecific antibodies can also be produced by chemical conjugation of two different mAbs. Other approaches include coupling of two parental antibodies with a hetero-bifunctional crosslinker, production of tandem single-chain Fv molecules, diabodies, bispecific diabodies, single-chain diabodies, and di-diabodies. In addition, a multivalent antibody construct comprising two Fab repeats in the heavy chain of an IgG and capable of binding four antigen molecules has been described (see PCT Publication No. WO 01/77342 and Miller et al. (2003) J. Immunol. 170(9):4854-61).
U.S. Pat. No. 7,612,181 (incorporated herein by reference in its entirety) provides a novel family of binding proteins capable of binding two or more antigens with high affinity, which are called dual variable domain binding proteins (DVD-Ig binding protein) or dual variable domain immunoglobulins (DVD-Ig). DVD-Ig molecules are binding proteins that may be used to bind two distinct epitopes on the same molecule or two different molecules simultaneously. DVD-Ig molecules are unique binding proteins comprised of two variable domains fused to N-terminal constant regions. The variable domains may be directly fused to one another or connected via synthetic peptide linkers of assorted length and amino acid composition. DVD-Ig binding proteins may be engineered with intact and functional Fc domains, or otherwise modified constant domains, allowing them to mediate appropriate effector functions and exhibit other desired properties. The DVD-Ig format, due to its flexibility of choice of variable domain pair, orientation of two antigen-binding domains, and the length of the linker that joins them, may provide novel therapeutic modalities.
There remains a need for constructs, including multispecific constructs, exhibiting better targeting, efficiency, and/or efficacy in binding to LRP-8, and/or improved transport and delivery of therapeutic agents across the blood brain barrier. Improved targeting of LRP-8 may lead to improvements in, e.g., preventing, diagnosing, and/or treating disorders such as brain disorders, neurological diseases, and/or brain cancers. Also, while a variety of structures have been provided in the art, with various advantages and disadvantages, new variable domain sequences can further improve the properties of binding proteins targeting LRP-8, or their cognate receptors.